In this proposal we aim to define the molecular mechanisms of division and differentiation in a phylum that consists entirely of bacterial species that live in osmotically stable, intracellular environments. During adaptation to intracellular life, microbes often exhibit a significant reduction in their genome size, resulting in the loss of metabolic and structural elements that are not required for life within a host cell. The bacterial cell wall, composed of peptidoglycan, protects most bacterial species from osmotic stress and is essential for cell division. Peptidoglycan also determines a bacterial cell?s shape, and by directing its synthesis and degradation microbes can effectively control cell size and differentiation between developmental forms. Nascent peptidoglycan biosynthesis is spatially and temporally restricted within bacterial cells via two known molecular complexes: the MreB complex, which is primarily associated with bacterial cell wall synthesis, and the FtsZ complex, which is associated with septal peptidoglycan synthesis required during cell division. Members of the Chlamydiae do not encode FtsZ and have long been thought to completely lack peptidoglycan. We recently discovered that several members of the Chlamydiaceae synthesize peptidoglycan but do not use it to form a canonical cell wall. Instead, these microbes utilize only septal peptidoglycan in their replicative forms, which is maintained, paradoxically, by an MreB complex. Here we propose a series of studies to investigate how members of the Chlamydiaceae temporally and spatially restrict peptidoglycan synthesis throughout the division process, efficiently controlling cell size, division, and the transition between developmental forms. Over the next five years we plan to increase our understanding of these fundamental processes by focusing on three major areas of investigation: 1) Identifying the mechanisms that direct and influence peptidoglycan synthesis and degradation in the absence of FtsZ, 2) characterizing polar localizing features present in Chlamydia and assessing their role in orienting peptidoglycan and the cell division complex, and 3) establishing the critical factors that influence bacterial cell size in an osmotically stable environment during the course of normal development and in response to cell stress. Genetically reduced microbes are attractive models for identifying the fundamental components of essential physiological processes. These planned studies will elucidate not only how genetically reduced microbes regulate cell size and divide in osmotically stable environments, but also illuminate the inherent versatility of the broadly conserved molecular complexes underlying these process.